Device and method for integrated diagnostics with multiple independent flow paths

ABSTRACT

Devices and methods for performing assays to determine the presence or quantity of a specific analyte of interest in a fluid sample. In devices according to this invention two separate flow paths are established sequentially in the device with a single user activation step. The first flow path delivers the analyte of interest (if present in the sample) and conjugate soluble binding reagents to the solid phase. If analyte is present, an analyte:conjugate complex is formed and immobilized. The volume of sample delivered by this first path is determined by the absorbent capacity of the solid phase, and not by the amount of sample added to the device, relieving the user from the necessity of measuring the sample. The sample/conjugate mixture is prevented from entering the second flow path because the capillarity and the surface energy of the second flow path prevent it from being wetted by this mixture. The second flow path allows a wash reagent to remove unbound conjugate and sample from the solid phase to the absorbant, and optionally to deliver detection reagents.  
     The invention may be adapted to many assay formats including, sandwich immunoassays, colloidal gold, or sol particle assays, heterogeneous generic capture assays and competitive assays.  
     In one embodiment, sandwich assays can be performed by immobilizing an analyte binding reagent on the solid phase, and drying a labeled analyte binding reagent in the first flow path. In a competitive assay embodiment, the first flow path would contain labeled analyte that is dissolved by the sample, and the analyte binding reagent is immobilized on the solid phase. In each of these embodiments, the assay can be further modified to run in a “generic capture” format, where the solid phase binding reagent is instead conjugated to a generic ligand such as biotin, and dried in the first flow path (either together or separately from the other assay reagents), and a generic ligand binding reagent (such as avidin) is immobilized on the solid phase.  
     Another aspect of the present invention includes a subassembly for the immunoassay device that is comprised of a plastic housing and a means for delivering fluid and/or wash solution. This subassembly comprises a structure formed from a hydrophobic polymer selectively treated with a water insoluble surface active agent that has been applied as a solution in an organic solvent rendering portions of the surface hydrophilic. When the surface is contacted with an aqueous liquid, it flows only along the treated areas, creating a defined fluid flow path, thereby delivering sample/conjugate solutions to said solid phase.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to devices and methods for performingassays to determine the presence or quantity of a specific analyte ofinterest in a fluid sample. Devices of this invention assay a measuredamount of sample employing at least two separate and distinct flow pathswhich are initiated simultaneously with a single user activation step.These paths are timed for the sequential delivery of assay reagents tothe reaction zone, followed by wash or substrate and wash reagents tothat zone. These inventive devices and methods may be used forqualitative, semi-quantitative and quantitative determinations of one ormultiple analytes in a single test format. They may be practiced withELISA, sol particle and other assay formats, and are particularlysuitable for simultaneous multiple analyte assays. These inventivedevices and methods provide for the controlled, self delivery ofreagents with no timed steps, and minimal user intervention, in mostinstances a single activation step.

[0003] 2. Background

[0004] Many prior art assay devices and systems require the user tomeasure or control the amount of sample added to the device, forexample, by dilution. Many prior art assays and systems also require theuser to perform a timed sequence of steps and/or to make multiplephysical interventions to the device in order to perform the assay.

[0005] Buechler et al., U.S. Pat. Nos. 5,458,852 and 5,885,527 (1995 and1999, respectively), disclose diagnostic devices which do not use porousmembranes. In assay methods using such devices, fluid flow isunidirectional and reaction and detection occur in distinct zones.Excess sample/conjugate mixture is used to wash the detection zone. Theuse of a separate, non-sample wash is not taught or suggested, limitingthe versatility of assays of this invention. Likewise, Buechler does notteach or suggest the creation of a second flow path. The time gate inBuechler functions as a delay mechanism in a fluid path, not to redirectfluid flow or to permit different fluids to flow sequentially through areaction zone. The methods and devices of Buechler et al. do not allowone to use multiple reagents, different wash and substrate reagents andcannot be used in an enzyme amplified assay.

[0006] Vonk, U.S. Pat. No., 5,185,127 discloses a filter stackcomprising a hydrophilic membrane (containing a binder for an analyte)above a hydrophobic membrane above an absorbent material. The membraneis impermeable to the sample but permeable to a wetting agent (e.g.acetone, surfactants, detergents, or alcohols, most preferably methanol)mixed with the sample. Sample is added to the device and trapped abovethe hydrophobic member. Thereafter the wetting agent is added, whichwhen mixed, allows the sample mixture to permeate into the absorbentmaterial. Wash and substrate may be added. Other disadvantages of thisdevice are that it (1) does not teach or suggest a defined fluid pathfor residual sample or wash, (2) does not permit controlled delivery ofpredetermined amounts of reaction components or wash, and (3) does notpermit sequential delivery of such components.

[0007] Clark, U.S. Pat. Nos. 5,726,010, 5,726,013 and 5,750,333 (1998)describes assay methods and devices that use the formation of a solidphase bound tertiary complex to detect an analyte of interest in a fluidsample. A key feature of Clark is the use of a reversible flow in achromatographic binding assay. An analyte-containing solution is appliedto the device and then is transported by capillary flow, first in onedirection and then in the opposite direction, along an elongated flowmatrix. The flow matrix includes four different regions. Region one iswhere the analyte-containing solution is mixed with a labeled antibody.Region two, also called the detection zone, contains the secondantibody, which is immobilized to a solid phase. Region three contains asite to apply a wash solution. Region four contains an absorbentreservoir located near region one and makes the flow go in the oppositedirection. A means to detect the presence or quantity of an analyte isalso included in the device. Clark does not automate steps. The usermust measure the sample volume, apply it to the device, monitor andcontrol timers, and physically activate the device. The mechanism forreversing flow does not allow for automated timing.

[0008] Assays and devices of the present invention overcome theshortcomings of the prior art and offer maximum results without usersample measurement or intervention beyond a single activation step.Assays of this invention may be particularly adaptable to situationswhere simultaneous detection of multiple analytes in a sample isdesirable. Assays of this invention are in a unit dose format, stable,capable of room temperature storage, reliable, easy to manufacture anduse, and available for a low cost per test. They have finally integratedpackaging for both liquid and dried reagents. In addition, the claimeddevices are self-timing for the delivery of reagents so there is minimaloperator involvement.

[0009] Definitions

[0010] Analyte—The molecule to be detected. For example, an analyte asused herein, may be a ligand, a single compound or a plurality ofcompounds that share at least one epitopic site to a receptor or anantibody.

[0011] Capillarity—The movement of a liquid in contact with a solid thatresults due to adhesive and cohesive forces and surface tension.Capillarity can be affected by the solid surface; the liquid surface, orboth.

[0012] Hydrophobic surface—Any surface not effectively wetted by wateror an aqueous sample.

[0013] Hydrophillic surface—Any surface wetted by water or an aqueoussample.

[0014] Conjugate soluble binding reagent—Reagent(s) deposited and driedon a solid support, i.e., the sample delivery channel, and have aspecific binding affinity for or chemical reactivity with the analyte ofinterest. Upon sample application, the conjugate soluble binding reagentbecomes dissolved and can begin to flow. Conjugate soluble bindingreagent can react with analyte, if present in the sample. Depending uponthe assay format, one conjugate soluble binding reagent can be labeledwith a detectable label and another with a binding reagent. Thesedifferent assay formats will be described in more detail below.

[0015] Solid phase zone—A material or a surface at the intersection ofat least two fluid flow paths.

[0016] Sample/conjugate mixture—The liquid mixture comprising the samplesolution and solubilized conjugate binding reagents.

[0017] Immobilized capture reagent—A molecule that is bound to a solidsupport and has a specific binding affinity for or chemical reactivitywith either the analyte of interest, or can be a receptor for one of theconjugate reagents e.g. avidin.

[0018] Sample delivery channel—The hydrophilic means where the conjugatesoluble binding reagents are dried and the means through which theliquid sample flows.

[0019] Socklet—The hydrophilic mesh material that contains the solidphase when a particulate solid phase is used.

[0020] Solid phase—The hydrophilic material to which the immobilizedcapture reagent is bound.

[0021] Second fluid path material—The hydrophobic material that providesthe secondary flow path. Examples of second fluid path material arebibulous material, plastic, or any other hydrophobic polymers. As thesurface active agent-containing wash solution flows through the secondfluid path material, the surface tension is reduced and allowssample/conjugate mixture to flow through the second fluid path material,which is now rendered hydrophilic.

[0022] Wash reagent—A liquid reagent that serves to remove unboundmaterial from the solid phase region. As used herein, the wash reagentcontains a surface active agent, such as a surfactant, or any othercomponent capable of allowing the wash to wet a hydrophobic surface.Some other examples of wash reagents are alcohol, e.g. methanol, or anyother water miscible organic solvents.

[0023] Substrate reagent—A liquid reagent that serves to facilitateanalyte detection by causing a detectable color reaction. A secondaryfunction of substrate reagent is to remove unbound material from thesolid phase region.

[0024] Wash/substrate reagent—In some embodiments of the presentinvention, the wash reagent may also contain a detection agent so that asingle solution is delivered via said second flow path to the solidphase zone.

[0025] Fluidic bridge—The fluid flow connection that is establishedbetween the socklet, second fluid path material and the absorbentreservoir. The fluidic bridge enables liquid flowing in the second flowpath to go into the absorbent reservoir.

[0026] Lance/wick—A component that is capable of piercing the seal ofthe liquid reagent containers, i.e., the wash and substrate reagentcontainers or the combined wash/substrate reagent container. The lancemay also include a wick, which facilitates the flow of the liquidreagents out of their storage containers and into the second fluid pathmaterial.

SUMMARY OF INVENTION

[0027] The present invention is directed to devices and methods forperforming an assay to determine the presence or quantity of a specificanalyte of interest in a fluid sample. Devices according to thisinvention comprise a solid phase capable of capturing the analyte ofinterest after washing unbound material from the solid phase. In oneaspect of the device two separate flow paths are establishedsequentially in the device with a single user activation step. The firstflow path delivers the analyte of interest (if present in the sample)and conjugate soluble binding reagents to the solid phase. If analyte ispresent, an analyte:conjugate complex is formed and immobilized.

[0028] The volume of sample delivered by this first path is determinedby the absorbent capacity of the solid phase, and not by the amount ofsample added to the device, relieving the user from the necessity ofmeasuring the sample. The sample/conjugate mixture is prevented fromentering the second flow path because the capillarity and the surfaceenergy of the second flow path prevent it from being wetted by thismixture.

[0029] The second flow path allows a wash reagent to remove unboundconjugate and sample from the solid phase to the absorbant, andoptionally to deliver detection reagents. The flow of wash reagent alongthe second flow path is initiated by user activation of the deviceimmediately after addition of sample to the first flow path. When washreagent migrates to the solid phase, and flows through and around it, itremoves unbound reagents and carries them to the absorbent. As a result,the incubation time of the sample and reagents in the solid phase isdetermined by the flow time of the wash reagent along the second flowpath, relieving the user of the requirement to intervene at a specifictime.

[0030] In one embodiment, the first flow path consists of a sample entryport, a filter element to remove particulates, an open capillary channelcontaining dried reagents, and a particulate solid phase. The secondflow path intersects the first, and consists of a wash reservoir, anabsorbent block, and a second fluid path material that extends from thereservoir, contacts the solid phase and finally contacts the absorbent.Sample is prevented from flowing into the second fluid path andabsorbent because the surface energy of the second fluid path materialis low relative to the sample surface tension, rendering it hydrophobic.The wash solution contains surfactant that allows it to wet the secondfluid path material, and thus allow the sample-binding reagent mixturefrom the solid phase to also flow into the second fluid path material.

[0031] The invention may be adapted to many assay formats including,sandwich immunoassays, colloidal gold, or sol particle assays,heterogeneous generic capture assays and competitive assays.

[0032] In one embodiment, sandwich assays can be performed byimmobilizing an analyte binding reagent on the solid phase, and drying alabeled analyte binding reagent in the first flow path. In a competitiveassay embodiment, the first flow path would contain labeled analyte thatis dissolved by the sample, and the analyte binding reagent isimmobilized on the solid phase. In each of these embodiments, the assaycan be further modified to run in a “generic capture” format, where thesolid phase binding reagent is instead conjugated to a generic ligandsuch as biotin, and dried in the first flow path (either together orseparately from the other assay reagents), and a generic ligand bindingreagent (such as avidin) is immobilized on the solid phase.

[0033] Another aspect of the present invention includes a subassemblyfor the immunoassay device that is comprised of a plastic housing and ameans for delivering fluid and/or wash solution. This subassemblycomprises a structure formed from a hydrophobic polymer. The hydrophobicpolymer has been selectively treated with a water insoluble surfaceactive agent that has been applied as a solution in an organic solventrendering portions of the surface hydrophilic. When the surface iscontacted with an aqueous liquid, it flows only along the treated areas,creating a defined fluid flow path, thereby delivering sample/conjugatesolutions to said solid phase.

[0034] Another aspect of the claimed device includes a solid phasesubassembly referred to as a socklet. The socklet is comprised of aparticulate solid phase material that is captured within a structurethat is permeable to the sample and liquid reagents, but which preventsthe particulate solid phase material from exiting the structure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1: Side view of one of the embodiments of the presentinvention.

[0036]FIG. 2: Exploded side view of the preferred embodiment of thepresent invention

[0037]FIG. 3: Side view schematic of first flow path of the preferredembodiment of the present invention.

[0038]FIG. 4: Schematic of second flow path of the preferred embodimentof the present invention.

[0039]FIG. 5: Schematic depicting some of the alternatives of thepresent invention for single or multiple analyte testing.

[0040]FIG. 6: Three-dimensional view of one of the embodiments of thepresent invention.

[0041]FIG. 7: Schematic depicting assay components for one of theembodiments of the present invention.

[0042]FIG. 8: Photographs of assay results using the embodiment ofExample 1.

[0043]FIG. 9: Schematic depicting another embodiment of the presentinvention.

[0044]FIG. 10: Schematic depicting another embodiment of the presentinvention.

LIST OF DEVICE COMPONENTS

[0045]81—Sample entry port of device incorporating embodiment of FIG. 9

[0046]82—Sample delivery/capillary channel of device incorporatingembodiment of FIG. 9

[0047]83—Holes through which wicks of device incorporating embodiment ofFIG. 9 are inserted

[0048]84—Hydrophobic exit channels of device incorporating embodiment ofFIG. 9

[0049]85—Hydrophilic solid phase material of device incorporatingembodiment of FIG. 9

[0050]86—Annular cup-shaped hydrophobic space of device incorporatingembodiment of FIG. 9

[0051]87—Hydrophobic reagent channels of device incorporating embodimentof FIG. 9

[0052]88—Hydrophobic channel of device incorporating embodiment of FIG.9

[0053]89—Array of capillary structures of device incorporatingembodiment of FIG. 9

[0054]90—Hydrophobic bibulous material of device incorporatingembodiment of FIG. 9

[0055]100—Sample entry port

[0056]101—Prefilter

[0057]102—Sample delivery channel

[0058]103—Conjugate (soluble) binding reagents

[0059]104—Socklet

[0060]105—Solid phase

[0061]106—Activator button

[0062]107—Die-cut hole in second fluid path material

[0063]108—Second fluid path material

[0064]109—Wash reagent

[0065]110—Substrate reagent

[0066]111—Absorbent

[0067]112—Wick

[0068]113—plastic housing

[0069]114A—Lance

[0070]114B—Lance

[0071]150—Hydrophobic bibulous material incorporating the embodiment ofFIG. 10

[0072]151—Sample entry port incorporating the embodiment of FIG. 10

[0073]152—Sample delivery channel incorporating the embodiment of FIG.10

[0074]153—Solid phase zone incorporating the embodiment of FIG. 10

[0075]154—Distal end zone incorporating the embodiment of FIG. 10

[0076]155—Absorbant incorporating the embodiment of FIG. 10

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] Assay Methods

[0078] This invention comprises diagnostic devices and methods to detectthe presence of at least one target analyte in a sample. FIG. 1 shows aside view of one of the preferred embodiments of device according tothis invention. Generally, the devices of the invention have thicknessesof about 1-30 mm, lengths of about 3-15 cm, widths of about 1-10 cm.Device size is determined by a large number of aesthetic, ergonomic, andperformance factors; for example width is affected by number of sampledelivery channels 102, length by time to result desired, and thicknessis affected by choice of reservoir and absorbent block materials. Thedevice depicted in FIG. 1 shows some of the general features of thedevices of the invention. The device comprises various elements: asample entry port 100, pre-filter 101, one or more sample deliverychannel(s) 102, socklet 104, solid phase 105, second fluid path material108, wash reservoir 109, substrate reservoir 110. Both the washreservoir 109 and the substrate reservoir 110 have lances 114A & B,which can serve as release mechanisms and wicks.

[0079] Referring to FIG. 1, the sample is added to the sample entry port100 and flows through a pre-filter 101. The sample entry port 100 can beany opening in the device housing for receiving sample and transferringit to the desired location for the start of the assay. Multiple sampledelivery channels 102 may also be incorporated into the device. Forexample, if one sample is being tested for the presence of multipleanalytes, once the sample is applied, the device is designed such thatequal aliquots are deposited in multiple sample delivery channels.Devices with multiple sample delivery channels will be discussed in moredetail below. Once the sample goes through the pre-filter 101, thesample flows into the hydrophilic sample delivery channel 102 wheredried conjugate soluble binding reagents 103 are located. When theliquid sample enters sample delivery channel 102 the conjugate solublebinding reagents 103 are dissolved into the sample solution andmobilized. Mixing the sample and conjugate allows the binding reactionsto begin. Depending on the assay format complexes may form betweenbinding reagents and analyte in the sample. These binding interactionscontinue to form while reagents continue to flow through the sampledelivery channel 102.

[0080] Once the sample/conjugate mixture reaches the distal end ofsample delivery channel 102, the sample/conjugate mixture flows to thesolid phase 105. This embodiment uses a socklet 104, a hydrophilic meshwhich holds a particulate solid phase material. The invention may bepracticed without a socklet, for example when non-particulate solidphase materials are chosen. FIG. 2 depicts the preferred embodimentusing socklet 104, which encases solid phase 105. The physicalproperties of the chosen flow materials, specifically their capillarityand surface energy ensure that the sample/conjugate mixture will bedrawn from sample delivery channel 102 into solid phase 105. Thesaturation of hydrophilic solid phase 105 allows the interaction betweenthe analyte (if present in the sample), conjugate soluble bindingreagents 103, (and preformed complexes of these elements), to combinewith immobilized capture reagent, which is irreversibly bound to solidphase 105. If the analyte of interest is present in the sample,conjugate soluble binding reagents-analyte of interest-immobilizedcapture reagent complex is formed and immobilized to solid phase 105.

[0081] Once the solid phase is saturated, flow within the first flowpath is completed, and flow stops because the solid phase 105 is encasedwithin a second fluid path material 108 which is not wetted by thesample. Second fluid path material 108 thus acts as a “barrier” to theflow of the sample, permitting the sample to only flow within andsaturate the first flow path but not enter the second flow path. Thissecond fluid path material 108 is a component of the second flow path.In this manner, the volume of sample tested by the device is preciselycontrolled by the volume of sample absorbed by solid phase 105. Sincethe device itself controls the volume of sample tested, the testoperator is relieved of the necessity of precision-pipeting the sample.

[0082] Saturation of solid phase 105 with sample containing conjugatesoluble binding reagent(s) 103 allows the aforementioned bindingreactions to occur. The time during which the binding reactions occur isdetermined by the time the sample mixture is allowed to reside withinsolid phase 105. The sample residence time is controlled by the arrivalof the wash solution 109 flowing within the second fluid path material108 of the second flow path.

[0083] The second flow path delivers wash reagent 109 and/or substratereagent 110 to the solid phase 105 in the following manner. Flow alongthe second flow path is initiated when the device is activated bypressing the activator button 106. Referring to FIG. 4, the wash reagent109 and substrate reagent 110 reservoirs are punctured by lances 114A &B and the wash and substrate reagents are simultaneously released. Thewash 109 and/or substrate 110 reagents flow into and through the secondfluid path material 108 towards the solid phase 105. Although the secondfluid path material 108 is initially hydrophobic, the wash reagent 109is able to wet and flow through this material 108 because the washreagent 109 contains a surface active agent which changes the surfacetension of the second fluid path material 108, “converting” the secondfluid path material 108 into a hydrophilic material, thus, allowing thewash reagent 109 to flow. When the wash reagent 109 reaches socklet 104,a portion of wash reagent 109 continues to flow along the second fluidpath material 108, past the socklet 104, through the wick 112 and intothe absorbant 111. In this manner, a flow bridge between the socklet 104and the absorbant 111 is established, utilizing the second fluid pathmaterial 108 now made hydrophillic by the wash solution. This fluidicbridge eliminates the “barrier” between the second flow path and socklet104 allowing sample and unbound reagents contained within socklet 104 toflow into absorbant 111. Once the second flow path is established,efficient washing of solid phase 105 occurs because wash reagent 109flows over, around, and through solid phase 105, and allows sample andunbound reagents within solid phase 105 to flow into absorbant 111.

[0084]FIG. 3 depicts the first flow path in more detail. The arrows showthe flow path after sample addition to sample entry port 100. FIG. 4depicts the second flow path in more detail. FIG. 4 is a side schematicview of the second flow path with arrows to depict fluid movements. Inthis embodiment, when the second fluid path material 108 is renderedhydrophilic, residual sample from the sample delivery channel 102 isdrawn into absorbent 111 because a portion of the second fluid pathmaterial 108 is in contact with sample delivery channel 102.

[0085] The interaction between the materials selected for the first andsecond flow paths is important for creating the second flow path. Oncethe boundary between the fluid paths is removed, absorbent block 111provides sufficient capillarity to pull the reagents into absorbent 111and sustain a separate second flow path that flows in a second directionfrom the first flow path. Delivering a liquid from this new directioncontrols the opening of the boundary between the flow paths.

[0086] The wash step is critical for the elimination of any unboundreagents to reduce non-specific binding and thereby increase assayperformance. This device of the claimed invention is designed to enablethe controlled delivery of a clean wash solution. In one preferredembodiment, this controlled delivery is dependent on the placement of(i.e., the distance between) solid phase 105 and the wash reservoir.

[0087] In another preferred embodiment, the device can be manufacturedin several different ways to engineer the opening of the boundarybetween the flow paths by the wash solution, without regard to theplacement of solid phase 105. For example, one may engineer a flow patharound the solid phase contact zone rather than underneath the solidphase. There are many ways to engineer the second flow path to ensurethat some wash/substrate reagent reaches the absorbent and therefore caninitiate sample washing.

[0088] If the conjugate soluble binding reagents 103 require asubstrate, e.g. if horse-radish peroxidase is used, the substratereagent follows the flow path of the wash reagent. The color thatdevelops at the solid phase is proportional to the level of analyte ofinterest present in the sample. Alternatively, the detector conjugatebinding reagent 103 used does not require a substrate e.g. conjugated tocolloidal gold or fluorescent tag, or an electrochemical detector. Inthis case, the substrate reservoir would not be included as part of thedevice.

[0089] In yet another alternative embodiment to FIG. 1 the device can bemodified to allow detection of the analyte on an electrochemical sensor.The solid phase 105 and socklet 104 are replaced by a set of electrodes(working, counter, and reference electrodes) printed in a recess on thesolid phase carrier part. Printed conductive pathways extend to the endof the device. Sample delivery channel 102 drains into a capillary spacecreated in the part over electrode (optionally containingcapillarity-inducing materials or structures). The hydrophobic bibulousmaterial 108 comprises the top of the space. The immobilized reagents103 are attached to the surface of the working electrode.

[0090] In this alternative embodiment to FIG. 1, sample applied to theentry port 100 migrates through the pre-filter 101 and along the sampledelivery channel 102, dissolving conjugate soluble binding reagents 103.The sample/conjugate mixture flows into the space above the electrodes,where flow stops and binding reactions take place. Activation of thedevice initiates flow of wash solution along the second fluid pathmaterial 108. When the wash reaches the area above the electrodes, thesample solution containing unbound analyte is drawn into the second flowpath and removed to the absorbant 111. Additional wash reagent 109 flowsthrough the space above the electrode, efficiently removing unboundreagents. If needed, a substrate reagent 110 flows sequentially alongthe second flow path, providing additional detection reagents. Theamount of bound analyte is detected electrochemically. The electrodescould be printed on an electrode cover part, and the first fluid pathwould pass through a die-cut hole 107 in the second flow path material108. Additionally, other detection methods that depend on a surfacecould be applied to this device. For example, if the parts were made oftransparent material, surface plasmon resonance could be measured on thebinding surface.

[0091] Device Components

[0092] Sample Entry Port

[0093] The sample entry port 100 is an opening in the plastic housingthrough which the sample is added. In one embodiment, sample entry port100 is located upstream of solid phase 105. There are different waysthat a sample may be applied to the invention device. One example of asample entry involves a cup structure comprising a raised surfacesurrounding the sample entry port provided on the top of the device. Inthis example, sample is dropped into the cup structure from a dispensingdevice, such as a piptettor or a syringe. The cup structure serves tocontain the sample around sample entry port 100. In another example, thesample may be applied to the device using a sample absorbing tip. Inthis example, a narrowed end of the device is adapted to directly absorbsample into the device. Liquid permeable fluid channels are providedwithin the device to transfer the sample from the tip of the device tosample delivery channels 102. The advantage of this example is that nodispensing device is required to aliquot sample. In this example, thetotal volume of channel structures and solid phase contained within thedevice controls the volume of sample required.

[0094] Pre-Filter

[0095] Sample entry port 100 may or may not contain pre-filter 101. Ifthe device of the present invention includes pre-filter 101, its primaryfunction is to remove any large particulate matter, which could clog thedevice and potentially interfere with the assay results. Pre-filter 101pores need not be small enough to remove blood cells if the remainder ofthe device is compatible with whole blood. However, if blood cells needto be removed, or additional separation is desired, the pre-filter 101used may be one available in the art for this purpose. In the preferredembodiment a single layer of hydrophilic medium is used.

[0096] In the preferred embodiment for multiple analyte testing, thesample moves through the pre-filter 101 into several sample deliverychannels 102. Each channel 102 contacts the pre-filter 101, but thechannels 102 are otherwise independent from each other. Thisindependence minimizes fluidic interaction and cross-contamination ofreagents between independent assays. Many alternatives are possible,depending on the type of assay desired.

[0097] Sample Delivery Channel

[0098] In the preferred embodiment, the first flow path consists of ahydrophilic sample delivery channel 102 that contains dried conjugatesoluble reagents 103. Although the preferred embodiments include driedconjugate soluble reagents in the sample delivery channel, someembodiments of the invention include the addition of conjugate solublereagent(s) to the sample before it is added to the device. The geometryand surface treatment of the sample delivery channel 102 are designed tocontrol the time of transit of the sample from pre-filter 101 to thesolid phase 105. For example, plastic capillary channels can be madehydrophilic by corona treatment or by treatment with a water-insolublesurfactant. The first flow path may also consist of other materials,including bibulous materials such as single or multiple conventionalimmunoassay strips containing dried conjugate binding reagents.

[0099] The sample delivery channel 102 may be designed to producesufficient capillarity to pull sample from the pre-filter into thesample delivery channel. In this case, the overall capillarity of eachof the components, pre-filter 101 and sample delivery channel 102 mustbe taken into account when designing the system so that the sample flowis in the direction of sample delivery channel 102. In this case, thesystem is active, in that fluid flow is determined by the physicalcharacteristics of the device and its components, rather than relying onan external force to induce fluid flow.

[0100] The primary functions of the sample delivery channel are to serveas a repository for conjugate binding reagents 103 and ensure that theyare distributed evenly throughout the sample, and to serve as a conduitfor the sample/reagent mixture to the solid phase. The design of sampledelivery channel 102 is important to provide even distribution of theconjugates. In some embodiments it may be desirable to promote mixing ofthe sample as it passes through the conjugate channel by incorporatingsurface topography into the parts.

[0101] In a generic capture system, the sample delivery channel may havetwo different reagents deposited and dried in the channel. The reagentsare a first immunological binding reagent to which a detectable labelhas been attached (the conjugate) and a second immunological reagent towhich a receptor molecule has been attached.

[0102] Solid Phase Material

[0103] The solid phase 105 contains a material coated with a reagent tobind the reaction products formed by the binding reagents and theanalyte. Solid phase materials may be glass fiber mats, non-wovensynthetic mats, sintered particulate structures, cast or extrudedmembrane materials, or other materials characterized by the presence ofadhesion within the material. The solid phase 105 may be a formed(molded or cast) open pore structure such as nylon or nitrocellulose.The solid phase 105 may also be a particulate material such as glassparticles or polymer particles. The solid phase 105 may also be asurface (such as an electrode or another sensing surface) with a definedfluid space bordered by the surface and the flow path material.

[0104] The solid phase materials may be slit, cut, die-cut or punchedinto a variety of shapes prior to incorporation into the device.Biological reagents may be applied to the materials before or afterforming the desired shape. Biological reagents may be attached to thematerials either passively or covalently. Examples of alternative shapesof the solid phase assemblies may be circular,square/rectangular-shaped, flattened ellipse shaped or triangularlyshaped.

[0105] In another embodiment, the solid phase 105 is a particulatematerial, which is not pre-formed into a structure that containsinternal adhesion. In this embodiment, the particulate solid phase 105is captured within a socklet. In this embodiment, the solid phasematerial is selected from the group consisting of inorganic or organicparticulates, glass beads, polymer particles. In the case of a sandwichimmunoassay, a preferred solid phase matrix is glass beads covalentlycoated and with avidin, which is the receptor-binding partner to abiotinylated binding reagent.

[0106] Socklet

[0107] Socklet 104 is a hydrophilic mesh material which physicallycaptures solid phase 105 without impeding the flow of liquids. In thisparticular embodiment, socklet 104 is permeable to the sample and liquidreagents, but does not allow the particulate solid phase material toexit the structure.

[0108] Second Fluid Path Material

[0109] In one embodiment, the second fluid path material 108 is definedby a hydrophobic medium that can be comprised of bibulous material,plastic, non-woven polyester fiber material, or any other hydrophobicstructure formed to provide a path for sample/reagent flow. In thepreferred embodiment, a porous second fluid path material 108 is placedin contact with a wash reservoir, extends to contact solid phase 105 andextends further to wick 112 and absorbent 111. The second fluid pathmaterial 108 surrounds the socklet 104 encased solid phase 105 (refer toFIG. 1). A die-cut hole 107 in second fluid path material 108 allowssample to travel from the sample delivery channel 102 to solid phase105. In the case of multiple solid phase 105 assemblies, the respectivenumber of die-cut holes 107 will be made in the second fluid pathmaterial 108 to allow delivery of the samples through the second fluidpath material 108 into solid phase 105.

[0110] In the preferred embodiment, the second fluid path material 108also serves as a fluid conduit for a detection reagent and isconstructed in such a way that it ensures that wash reagent 109 flowsbefore substrate reagent 110.

[0111] Absorbent

[0112] Absorbent 111, serves as an excess reagent reservoir that iscapable of accommodating a volume of liquid in excess of the totalsample volume and the total volume of all other liquid reagents. Inalternative embodiments, the excess reagent reservoir can be a moldedvessel for holding excess fluid or a continuation of the second fluidpath material that has been folded or otherwise made into an excessreagent reservoir.

[0113] Reagent System

[0114] The conjugate soluble binding reagents 103 are dried into sampledelivery channel 102, and are dissolved by the sample. In the example ofa sandwich immunoassay, the preferred reagents include first and secondconjugate soluble binding reagents 103. A first conjugate solublebinding reagent 103 may be an antibody (or antigen) conjugated to adetectable label. The detectable label may be a light absorbing particlesuch as colloidal gold or a colored latex particle, a phosphorescentmolecule, a fluorescent molecule or an enzyme, such as horseradishperoxidase. Additionally, the detectable signal can be electrochemicalor any other signal used in the art as an assay readout. The secondconjugate soluble binding reagent 103 may be an antibody (or antigen)conjugated to a receptor molecule such as biotin. Alternatively, thebinding partners for the assays described herein can include antigens,antibodies, ligands, receptors, nucleic acid molecules, chemicalreactants, fragments of these, and other such reagents used in the artfor conducting assays.

[0115] The concentration of conjugate soluble binding reagent(s) 103 inthe sample is affected by the rate of dissolution of the conjugatesoluble binding reagent(s) 103 by the sample, the flow rate of thesample, and the by amount of conjugate soluble binding reagent(s) 103applied to sample delivery channel 102.

[0116] Further embodiments of the described invention include the assayof enzymatic or chemical reactions by immoblizing a reactant orsubstrate on solid phase 105, and detecting the product, or bydiffusively or non-diffusively immobilizing the reactant or substrate inthe first flow path, and detecting the product in the solid phase zone.

[0117] Different Embodiments of the Present Invention

[0118] In another embodiment, depicted in FIG. 9, the first fluid pathconsists of a sample entry port 81 in fluidic communication with each oftwo hydrophilic sample delivery/capillary channels 82 containing driedbinding reagents. The distal end of each channel 82 is in fluidcommunication with the underside of the hydrophilic solid phase material85.

[0119] The second fluid path begins in a piece of hydrophobic bibulousmaterial 90 that is butted against an array of capillary structures 89which withdraw wash reagent from hydrophobic bibulous 90 intohydrophobic channel 88. This channel splits into two hydrophobic reagentchannels 87, which connect to an annular cup-shaped hydrophobic space 86around the solid phase 85. Connected to the distal side of this space isa set of hydrophobic exit channels 84 which lead to wicks inserted intoholes 83. The wicks are in fluidic communication with an absorbent blockunder the device (not shown).

[0120] In operation, sample enters the port 81, flows down the channel82 and dissolves the dried reagents. Flow continues down the channel andinto the solid phase 85, where flow stops when the solid phase 85 issaturated. The flow of the sample/reagent mixture does not flow into thesecond flow path because the surfaces of the second flow path arehydrophobic.

[0121] Coincident with addition of sample, the device is activated,initiating flow of wash along bibulous material 90. When the washreaches the array of capillary structures 89, it migrates from thehydrophobic bibulous material 90 and into the hydrophobic channel 88.Flow of the wash splits and continues along reagent channels 87. Whenthe flow reaches annular cup-shaped hydrophobic space 86, a portion ofthe wash passes around solid phase 85 and into the exit channels 84.Wetting of these surfaces by the wash renders them hydrophilic, and thesample in the solid phase 85 is then drawn into the exit channels 84 andinto the wicks 80, followed by the remaining wash. In the case of anenzyme amplified assay, substrate solution would flow sequentially afterthe wash along the second fluid path.

[0122] When the label of one of the conjugate soluble binding reagentsis an enzyme, the substrate flows from a reservoir along the hydrophobicbibulous material 90, following the wash reagent. The substrate fluidpath follows the wash fluid path, and therefore is part of the secondflow path. The assay is complete when the enzyme label in the solidphase 85 has reacted with substrate.

[0123] In another embodiment of the invention depicted in FIG. 10, allof the flow paths are created on a single piece of hydrophobic bibulousmaterial 150. As shown in FIG. 10, a strip of hydrophobic bibulousmaterial 150 is printed with a water insoluble surfactant to create afirst hydrophilic fluid path consisting of a sample entry port 151, asample delivery channel 152, and a solid phase zone 153. Sample isapplied to sample entry port 151, migrates along the first fluid path,and dissolves conjugate soluble binding reagents deposited in sampledelivery channel 152. When sample/conjugate mixture reaches solid phasezone 153, the analyte in the sample and analyte/reagent complexes bindto the immobilized reagent attached to the solid phase zone 153. Whensolid phase zone 153 (as defined by the limits of application of thewater-insoluble surfactant) is saturated, flow stops. Wash reagentcontaining surfactant is applied (by the operator or by methodsdescribed in previous examples) to the distal end zone 154 and migratesalong bibulous material 150 rendering it hydrophilic. When the washreaches solid phase zone 153, it allows the sample solution in solidphase zone 153 to migrate through bibulous material 150, and intoabsorbant 155. Additional wash migrates through solid phase zone 153,removing all unbound reagents. If a substrate reagent is required, it isapplied to distal end zone 154 after the wash, and flows alongsequentially after the wash.

[0124] Alternatively, this fluid path in this embodiment can be cut froma sheet of hydrophilic bibulous material and bonded to hydrophobicbibulous material 150. The underside of bibulous material 150 iscontacted with hydrophilic absorbant 155.

[0125] Alternative Structures for Single or Multiple Analyte Testing

[0126]FIG. 5A, depicts a single sample entry port 100, single sampledelivery channel 102 and a single solid phase 105 assembly. Thisstructure is useful to test a single analyte per sample. No controls arepresent in this format. Multiple units of this structure may be placedwithin a single device to perform a single test on multiple samples. Theuse of multiple channels is very versatile. For example, one coulddesign a colloidal gold sandwich assay side-by-side with an enzymeamplified competitive assay. Alternatively, channels may be used forpositive and negative controls.

[0127]FIG. 5B, shows a structure with a single sample entry port 100,two separate sample delivery channels 102 and two separate solid phase105 assemblies. It can be used for testing a single sample for two ormore unique analytes or a single sample for a single analyte and acontrol. Different assay formats may be used simultaneously with thisstructure. Multiple units of this structure may be incorporated into asingle device to test multiple samples for multiple analytes.

[0128]FIG. 5C, is an example of a structure with a single sample entryport 100, single conjugate channel 102 and two separate solid phase 105assemblies. This structure is most useful for use in an immunoassayformat where the biological reagent present in the two solid phase 105assemblies are different, particularly when one solid phase 105 acts asa control for the other.

[0129] Operation of the Device

[0130]FIG. 6 depicts one embodiment of the claimed invention device. Theuser adds to the sample entry port 100 a volume of sample at leastsufficient to fill the sample delivery channels 102, prefilter 101, andsolid phase 105 (about 30 microliters in the examples) and pressesactivator button 106 to activate the device. The activation processcauses the lance/wick to rupture seals on the wash 109 and substrate 110containers, initiating flow of these reagents. The sample flows throughthe filter element 101, and into the sample delivery channel 102. Thesample dissolves the dried conjugate soluble binding reagents 103,initiating the reaction. The reaction mixture flows into solid phase105, where the second phase of the binding reaction occurs i.e.,immobilization of analyte-conjugate soluble binding reagents complex tosolid phase 105. The first flow path stops at the distal end of thesolid phase. The incubation time in the solid phase 105 is determined bythe transit time of the wash solution 109 along the second fluid pathmaterial 108. Wash reagent 109 removes the unbound reactants from thesolid phase 105, and substrate 110 flowing sequentially after the washsolution 109 then develops a signal.

[0131] Optional Embodiments

[0132] Other Labels

[0133] When the label used is a colored particle e.g., colloidal gold,or a fluorescent or electrochemical tag, a substrate is not required,and the assay is complete after the wash has removed unbound materialfrom the solid phase binding zone.

[0134] Semi-Quantitative Assays

[0135] In a semi-quantitative assay, it is sometimes desirable tocompensate for device and sample variations by comparing the analyteassay signal against that generated in a control reaction. In this case,reagents for a test reaction and a control reaction may be driedtogether in the sample delivery channel. In this embodiment, the sampledelivery channel is then split, feeding two solid phase binding zones,each specific for one of the reactions.

[0136] Quantitative Assays

[0137] In a quantitative assay, precision and enhanced sensitivity maybe achieved by precisely controlling the timing steps of the reaction.The precision of this assay device may be improved by developing aninstrument that controls the time that the sample resides in the sampledelivery channel, the time the sample/reagent mixture resides in thesolid phase, and the time after washing that the assay is read.

[0138] Experimental Procedures

EXAMPLE 1

[0139] This example relates to the present methods and devices for thedetection of canine heartworm antigen in a sample.

[0140] Preparation of Anti-Heartworm-Enzyme Conjugate:

[0141] Horseradish peroxidase (HRP) (20 mg) was oxidized with a 100-foldmolar equivalent of sodium periodate at pH 4.5, then mixed with 1.25equivalent amount by weight of affinity-purified chicken-anti-heartwormantibody. After 1.5 hours of reaction, the HRP-antibody conjugate wasstabilized by reduction with a 100-fold molar equivalent of sodiumborohydride, incubated for a further 0.5 hours. Conjugate was stored at1 mg/ml protein concentration in conjugate diluent. This HRP-antibodyconjugate is one of the conjugate soluble binding reagents that will bedried in the sample delivery channel of the device prior to use.

[0142] Preparation of Biotinylated Anti-Heartworm Antibody:

[0143] Affinity-purified rabbit-anti-heartworm antibody (20 mg) wasreacted at alkaline pH with 15 molar equivalents ofbiotin-amidocaproyl-NHS ester. Unreacted biotin was separated bydialysis. Antibody biotinylation may be determined using standardmethods such as avidin binding. This biotinylated antibody is the secondconjugate soluble binding reagent that will be dried in the sampledelivery channel of the device prior to use.

[0144] Preparation of Solid Phase:

[0145] 1.4 kg of borosilicate glass spheres (65μ±10μ diameter) werestirred for 4 hours with 1.4 L of aqueous 1% aminopropyltriethoxysilane(1 w/v (kg/L) to produce aminopropyl-glass. After washing over acoarse-porosity sintered glass funnel with 5 L of water and curing at50C overnight, 600 g of the aminopropyl-glass was stirred with 50 mL of4.4% solids carboxyl-latex (87 nm diameter, 430 μeq/g) in 600 mL of 50mM barbituric acid buffer, pH 3.3. The latex was coated to theaminopropyl-glass by stirring 40 mg/mL aqueous EDAC into the glasssuspension in two additions of 10 mL spaced 30 minutes apart, thenallowed to react for an additional hour. Unbound latex was removed fromthe resulting latex-coated glass by washing with 2 L of 0.2 M NaClfollowed by 8 L of water. To coat the latex-glass with neutravidin, thewashed latex-coated glass (600 g) was suspended in 600 mL of 50 mM MES(2-N-(morpholinoethanesulfonic acid)) buffer, pH 6.0 and stirred with 10mL of 29 mg/mL aqueous neutravidin followed by two additions of 14.5 mLof 40 mg/mL aqueous EDAC spaced 30 minutes apart, then allowed to reactfor an additional hour. Unbound neutravidin was removed from theresulting neutravidin-latex-glass by washing with 2L of buffer A (50 mMpotassium phosphate, 0.1% EDTA, pH 8.0) containing 0.1% Tween 20,followed by 2 L of buffer A containing 0.5 M NaCl, and finally 2L ofbuffer A.

[0146] The described solid phase has several advantages. The pore sizedetermined by the glass particles provides high capillarity whileallowing easy flow of blood cells without entrapment. Coating the glasswith latex particles increases the surface area, allowing greateramounts of immobilized reagents and thus faster binding reactions. Thelatex also reduces non-specific binding and provides a surface thatallows easily coupling of reagents. Finally, the raw materials areinexpensive, readily available, and allow the preparation of the finalsolid phase in bulk process.

[0147] The assay device was assembled as follows (Refer to FIG. 1):

[0148] 1. Depositing 5.5 μl of a slurry of the solid phase 105 into eachof two spots on a solid phase carrier and drying to a solid.

[0149] 2. A pre-formed nylon mesh socklet (Saatitech PA-3121) 104 washeat-staked over the dried solid phase 105.

[0150] 3. A strip of hydrophobic non-woven polyester (Ahlstrom 6627) 108with two 0.093″ holes was heat-staked over the solid phase 105. Thishydrophobic material serves as the second fluid path material 108 of thedevice.

[0151] 4. Each sample delivery channel 102 was made hydrophilic bydeposition of 680 nl of a 2.5% solution of Brij 72 surfactant indiacetone alcohol using a BioDot QuantiBiojet.

[0152] 5. The parts were dried and 212 nl of 50% sucrose were depositedon top of the surfactant layer followed by drying.

[0153] 6. The chicken anti-heartworm-enzyme conjugate (100 nl, 1 mg/ml)and the biotinylated rabbit anti-heartworm antibody (50 nl, 1 mg/ml)both serving as conjugate sluble binding reagents 103, were depositedinto a portion of the sample delivery channel 102 of the part from step5 and dried.

[0154] 7. A glass fiber pre-filter (Ahlstrom 8980) 101 was placed overthe sample entry port 100, and a plastic cover was ultrasonically weldedover the deposited antibodies to make the sample delivery channelassembly 102.

[0155] 8. The solid phase assembly 105 and the sample delivery channelassembly 102 were attached to each other by heat staking.

[0156] 9. Absorbent wicks (Filtrona R-14545, bondedpolyester/polyethylene, 0.078″ dia.) were placed in the holes of thedevice, and an absorbent cellulose acetate block 111 was held againstthese wicks.

[0157] A serum sample containing canine heartworm antigen was added tothe pre-filter of the assembled device. The sample flowed into thesample delivery channels and dissolved the deposited antibodies,facilitating the reaction of the antibodies with the antigen. Thereaction mixture flowed further down the sample delivery channel andinto the solid phase, where flow stopped. Biotinylated antibodies in thereaction mixture bound to the solid phase, capturing immune complexeswith the antigen and antibody-enzyme conjugate. FIG. 9 depicts theimmuno-complex formed in this experiment.

[0158] Coincident with the addition of the sample, the end of the stripof hydrophobic polyester is placed in contact with a reservoircontaining 250 μl of a surfactant-containing wash reagent containing0.025 M citrate, 0.15 M NaCl, 0.5% Kathon, 1% Triton X-100. 0.75% Drymilk, 1.5% BSA (from 30% liquid stock). The wash reagent is made to pH7.0 and filtered through a 0.2 micron filter. The wash reagent startsmigrating through the strip of hydrophobic polyester. When the washreagent reaches the region of the strip of hydrophobic polyesteradjacent to the solid phase, it migrates into the solid phase, removingunbound antibodies. The wash continues to migrate through the strip ofhydrophobic polyester and into the absorbent wicks and block, bringingthe antibody solution along. When the wash reservoir is empty, the stripof hydrophobic polyester is placed in contact with a second reservoircontaining 250 μl of TMB substrate solution (Moss, TMBM 1000), whichmigrates through the strip following the wash. When the substratereaches the solid phase, it replaces the wash solution and a blue colordevelops in the solid phase in proportion to the amount of boundantibody-enzyme conjugate. When a similar device was run using anegative serum sample, no visible color developed. See FIG. 10. FLOWTIMES Sample to solid phase 10-15 seconds Wash to solid phase 30 secondsSample removal complete 1 minute 45 seconds Color visible for sample 3minutes for strong positive containing analyte 6 minutes for weakpositive

EXAMPLE 2

[0159] The device of example 1 can be simplified by use of a fluorescentlabel or colored particle e.g. colloidal gold, attached to the chickenanti-heartworm antibody instead of using HRP as the conjugate. In thiscase, the substrate reagent can be omitted, and the signal is viewedafter the unbound reagent is washed away.

EXAMPLE 3

[0160] To detect multiple analytes in a single sample, a multi-channeldevice is constructed from the device in example 1 by feeding multiplesample delivery channels from a single sample entry port (FIG. 7). Eachsample delivery channel will contain different conjugate bindingreagents to detect the different analytes. Thus, four solid-phasecontainers are constructed to be adjacent to each other. The assembly isattached to a plastic part with four sample delivery channels.

What is claimed is:
 1. A device for performing an assay which determinesthe presence or quantity of an analyte in a fluid sample (1) using asolid phase capable of capturing said analyte or the analyte bound to abinding partner for the analyte, and (2) detecting the binding of saidanalyte or said analyte bound to its binding partner after washingunbound material from said solid phase, said device comprising: (a) afirst flow path for sample entry and delivery to a solid phase (d),which is independent of a second flow path defined by (b), a secondfluid path material; (b) a second fluid path material essentiallyimpermeable to said liquid sample but which defines a second flow pathfor said sample when made permeable by contact with a wash reagent (c);(c) a wash reagent permeable to said second fluid path material, whichwhen released flows through said second fluid path material; (d) a solidphase zone permeable to said liquid sample, at the intersection betweensaid first and second flow paths, whereby said liquid sample initiallyflows only along said first flow path and said solid phase zone intosaid solid phase but not into said second fluid path material until saidsecond fluid path material is rendered hydrophilic by said wash reagent;(e) a means to test a single sample for a single analyte or a means totest a single sample for multiple analytes.
 2. Device of claim 1 whereinfirst flow path is through a capillary space called a sample deliverychannel.
 3. Device of claim 1 wherein first flow path flows along abibulous material.
 4. Device of claim 1 wherein first flow path isthrough a sample delivery channel containing dried conjugate solublebinding reagents.
 5. Device of claim 1 wherein binding partners for theassays can include antigens, antibodies, ligands, receptors, nucleicacid molecules, chemical reactants, fragments of these, and any othersuch reagent used in the art for conducting assays.
 6. Device of claim 1wherein conjugate soluble binding reagents are dried in the sampledelivery channel.
 7. Device of claim 1 wherein at least one of theconjugate soluble binding reagents is added to the sample prior toadding said sample to said device.
 8. Device of claim 1 wherein theassay involves an immobilized reactant or substrate on the solid phase,and detection occurs on said solid phase.
 9. Device of claim 1 whereinthe assay involves having the reactant or substrate in said first flowpath and detection occurs on said solid phase.
 10. Device of claim 1wherein the solid phase is selected from the group consisting of: glassfiber mats, non-woven synthetic mats, sintered particulate structures,cast or extruded membrane materials, materials characterized by thepresence of adhesion within the material, molded or cast open porestructure such as nylon or nitrocellulose, particulate material such asglass particles or polymer particles, or an electrochemical detector.11. Device of claim 1 wherein wash and/or substrate reagent(s), orwash/substrate reagent is delivered from a pierceable container(s)following activation of said device.
 12. Device of claim 1 whereinabsorbent block is comprised of an absorbent material independent ofsecond fluid path material.
 13. Device of claim 1 wherein second flowpath may be through bibulous material or plastic.
 14. Device of claim 1wherein the sample entry port may be located upstream of the solid phaseor directly above the solid phase and sample may be applied via a cupstructure or a sample absorbing tip.
 15. A device for performing anassay which determines the presence or quantity of an analyte in a fluidsample (1) using a solid phase capable of capturing said analyte or theanalyte bound to a binding partner for the analyte, and (2) using atleast one diffusibly immobilized capture reagent capable of binding saidanalyte or an analogue of said analyte, at least one substrate reagentor at least one combined wash/substrate reagent, and (3) detecting thebinding of said analyte or said analogue of said analyte after washingunbound material from said solid phase, said device comprising: (a) asample entry port in fluid communication with a first flow path (b); (b)a first flow path in fluid communication with said sample entry port ona first end, having a solid phase containing at least one immobilizedreagent therein, and a second fluid path material, on a second end, (c)a second fluid path material which is initially impermeable to saidliquid sample, but which is made permeable to said sample when saidsecond fluid path material is contacted with wash, or said combinedwash/substrate reagent, or wash followed by substrate reagent; (d) asecond flow path for said sample, independent of said first flow path,said second path having a first end and an excess reagent reservoir atsecond end, with said solid phase therein, said second flow path definedphysically by the second fluid path material and fluidically by whensaid wash or said combined wash/substrate reagent renders said secondfluid path material permeable to said sample, and said second flow pathdelivers wash and or said combined wash/substrate reagent to and throughsaid solid phase, thereby removing unbound materials from said solidphase and delivering said wash or said wash/substrate reagent to saidsolid phase; (e) a means to test a single sample for a single analyte ora means to test a single sample for multiple analytes.
 16. Device ofclaim 15 wherein first flow path is a capillary space called a sampledelivery channel.
 17. Device of claim 15 wherein first flow path movesalong a bibulous material.
 18. Device of claim 15 wherein first flowpath is a sample delivery channel containing dried reagents.
 19. Deviceof claim 15 wherein at least one of the conjugate soluble bindingreagents is added to the sample prior to adding said sample to saiddevice.
 20. Device of claim 15 wherein binding partners for the assayscan include antigens, antibodies, ligands, receptors, nucleic acidmolecules, chemical reactants, fragments of these, and any other reagentused in the art for conducting assays.
 21. Device of claim 15 whereinthe sample entry port may be located upstream of the solid phase ordirectly above the solid phase and sample may be applied via a cupstructure or a sample absorbing tip.
 22. Device of claim 15 wherein saidwash reagent, wash/substrate reagent, or wash and substrate reagents aredelivered from pierceable containers following activation of saiddevice.
 23. Device of claim 15 wherein absorbent block comprises anabsorbent material independent of second fluid path material.
 24. Deviceof claim 15 wherein second flow path may be bibulous material orplastic.
 25. A device of claim 15 wherein the solid phase material isselected from the group consisting of glass fiber mats, non-wovensynthetic polymer mats, sintered particulate structures, cast orextruded membrane materials, or other materials characterized by thepresence of adhesion within the material, or molded or cast open porestructure such as nylon or nitrocellulose, or particulate material suchaas glass particles or polymer particles, or an electrochemical detector.26. A device of claim 15 which includes said assay wherein detectionoccurs via immobilized reactant or substrate on solid phase, or viareactant or substrate present in first flow path that flows into saidsolid phase where detection occurs.
 27. A solid phase subassemblycomprising a particulate solid phase material physically captured withina socklet which is permeable to sample and liquid reagents, but whichdoes not allow the particulate solid phase material to exit saidstructure.
 28. A method for performing an assay which determines thepresence or quantity of an analyte in a fluid sample (1) using a solidphase capable of capturing said analyte or analyte bound to a bindingpartner for the analyte, and (2) detecting the binding of said analyteor said analyte bound to its binding partner after washing unboundmaterial from said solid phase, said method comprising: (a) contacting aliquid sample with a first flow path, (in the sample delivery channel)said path in fluid communication with a sample entry port on a firstend, a solid phase containing at least one immobilized reagent therein,said solid phase within said first flow path, and a second fluid pathmaterial, on a second end, said second fluid path material incommunication with said solid phase; (b) flowing said liquid sampleinitially through said first flow path to said second fluid pathmaterial which is initially impermeable to said liquid sample, but whichis made permeable to said sample when said second fluid path material iscontacted with wash reagent, substrate reagent or combinedwash/substrate reagent, said second fluid path material to define asecond flow path for said sample, independent of said first flow path,said second flow path having a first end and an excess reagent reservoirat a second end, with said solid phase therein, which second flow pathdelivers said wash reagent, or wash and substrate reagents, or combinedwash/substrate reagent to and through said solid phase, thereby removingunbound materials from said solid phase, and delivering wash orwash/substrate reagent to said solid phase; (c) determining the presenceor amount of analyte using the analyte detection reagent.
 29. Method ofclaim 28 wherein said binding partners for the assays can includeantigens, antibodies, ligands, receptors, nucleic acid molecules,chemical reactants, fragments of these, and any other reagent used inthe art for conducting assays.
 30. An assay method for the determinationof a analyte in a liquid sample comprising the steps of: (a) forming amixture of a sample containing the analyte with: (i) a firstimmunological binding partner to the analyte, wherein the firstimmunological binding partner contains a detectable label, and (ii) asecond immunological binding partner of the analyte wherein the secondimmunological binding partner has been modified by the covalentattachment of a receptor molecule; (b) allowing the first and secondimmunological binding partner to form an immune complex with theanalyte; (c) contacting the mixture in (a) with a liquid permeable solidphase material comprising a binding partner for said receptor whichreceptor has been covalently attached to said second immunologicalbinding partner, and which said binding partner is non-diffusively boundto said solid phase, said solid phase in contact with a second fluidpath material initially impermeable to said sample, and which defines aflow path; (d) allowing the receptor molecule attached to the secondimmunological binding partner to form a complex with thereceptor-binding partner non-diffusively bound to the solid phasematerial; (e) flowing a liquid reagent permeable to said second fluidpath material along a second flow path, within said second fluid pathmaterial, such that said liquid reagent removes unbound reactants fromsaid solid phase; (f) detecting the label attached to the firstimmunological binding partner as part of the immune complex formed in(b).
 31. Method of claim 30 wherein the binding partners can includeantigens, antibodies, ligands, receptors, nucleic acid molecules,chemical reactants, fragments of these, and other such reagents used inthe art for conducting assays.
 32. Method of claim 30 wherein thedetecting step (f) is by a flowing a substrate reagent through saidsecond flow path in said second fluid path material.
 33. Method of claim30 wherein said substrate reagent is a substrate for an enzyme. 34.Method of claim 30 wherein said reagent permeable to said second fluidpath material is an aqueous solution containing a surface active agent.35. Method of claim 30 wherein said surface active agent is asurfactant, alcohol, or any other miscible organic solvent.
 36. Methodof claim 30 wherein said second fluid path material is a bibulousmaterial.
 37. Method of claim 36 wherein said bibulous material is anon-woven polyester fiber material.
 38. Method of claim 30 wherein saidfirst or second immunological binding partner to said analyte is addedto the sample prior to initiation of said assay.
 39. Method of claim 30wherein said second fluid path material is plastic.
 40. Method of claim39 wherein said second fluid path material is a structure formed from ahydrophobic polymer.
 41. Method of claim 30 wherein said solid phase isa formed (molded or cast) in an open pore structure.
 42. Method of claim41 wherein said open pore structure is nylon or nitrocellulose. 43.Method of claim 30 wherein said solid phase is an electrochemicaldetector.
 44. Method of claim 41 wherein said solid phase is aparticulate material.
 45. Method of claim 44 wherein said solid phase isglass particles.
 46. Method of claim 44 wherein said solid phase ispolymer particles.
 47. Method of claim 30 wherein said detectable labelof conjugate binding reagent is a light absorbing particle.
 48. Methodof claim 47 wherein said light absorbing particle is colloidal gold. 49.Method of claim 47 wherein said light absorbing particle is a coloredlatex particle.
 50. Method of claim 47 wherein said detectable label isa phosphorescent molecule.
 51. Method of claim 30 wherein saiddetectable label is a fluorescent molecule.
 52. Method of claim 30wherein said detectable label is an enzyme.
 53. Method of claim 52wherein said enzyme is horseradish peroxidase.
 54. Method of claim 30wherein said receptor molecule covalently attached to said secondconjugate binding partner is biotin.
 55. Method of claim 54 wherein saidreceptor-binding partner non-diffusively bound to said solid phase isavidin.
 56. Device of claim 44 or 45 wherein said solid phase materialis selected from the group consisting of inorganic or organicparticulates, glass beads, or polymer particles.
 57. Method of claim 30wherein detection occurs via immobilized reactant or substrate on saidsolid phase, or via reactant or substrate present in first flow pathinto said solid phase where detection occurs.
 58. An assay method forthe determination of an analyte in a liquid sample comprising the stepsof: (a) forming a mixture of a sample containing the analyte with: (i) afirst conjugate binding partner to the analyte, wherein the firstconjugate binding partner contains a detectable label, (ii) a secondconjugate binding partner of the analyte wherein the second conjugatebinding partner has been modified by the covalent attachment of areceptor molecule, and (iii) a solid phase material wherein a bindingpartner of the receptor molecule attached to the second conjugatebinding partner has been non-diffusively bound to the solid phasematerial, (b) simultaneously allowing the first and second conjugatebinding partners to form an immune complex with the analyte, and thereceptor molecule attached to the second conjugate soluble bindingpartner and the receptor-binding partner non-diffusively bound to thesolid phase to form an immobilization complex, wherein said solid phaseis in contact with a second fluid path material initially impermeable tosaid mixture, and which defines a first flow path; (c) flowing a washreagent, or wash/substrate reagent containing a surface active agentalong a second flow path, within said second fluid path material, suchthat the surface active agent allows the wash reagent to flow throughsaid second flow path, thereby removing unbound reactants from saidsolid phase; (d) detecting the label attached to the first conjugatebinding partner as part of the immune and immobilization complexesformed in (b).
 59. Method of claim 58 wherein the detecting step (d) isby flowing a substrate reagent through said second flow path in saidsecond fluid path material.
 60. Method of claim 59 wherein saidsubstrate reagent is a substrate for an enzyme.
 61. Method of claim 58wherein said reagent permeable to said second fluid path material is awash solution containing a surface active agent.
 62. Method of claim 58wherein said surface active agent is a surfactant, alcohol, or any othermiscible organic solvent.
 63. Method of claim 58 wherein said secondfluid path material is a bibulous material.
 64. Method of claim 63wherein said bibulous material is a non-woven polyester fiber material.65. Method of claim 58 wherein said second fluid path material isplastic.
 66. Method of claim 58 wherein said second fluid path materialis a structure formed from a hydrophobic polymer.
 67. Method of claim 58wherein said solid phase is a formed (molded or cast) open porestructure.
 68. Method of claim 67 wherein said open pore structure isnylon.
 69. Method of claim 67 wherein said open pore structure isnitrocellulose.
 70. Method of claim 58 wherein said solid phase is aparticulate material.
 71. Method of claim 58 wherein said solid phase isglass particles.
 72. Method of claim 58 wherein said solid phase ispolymer particles.
 73. Method of claim 58 wherein said detectable labelis a light absorbing particle.
 74. Method of claim 73 wherein said lightabsorbing particle is colloidal gold.
 75. Method of claim 73 whereinsaid light absorbing particle is a colored latex particle.
 76. Method ofclaim 58 wherein said detectable label is a phosphorescent molecule. 77.Method of claim 58 wherein said detectable label is a fluorescentmolecule.
 78. Method of claim 58 wherein said detectable label is anenzyme.
 79. Method of claim 78 wherein said enzyme is horseradishperoxidase.
 80. Method of claim 58 wherein said receptor moleculecovalently attached to said second conjugate binding partner is biotin.81. Method of claim 80 wherein said receptor-binding partnernon-diffusively bound to said solid phase is avidin.
 82. Method of claim58 wherein detection occurs via immobilized reactant or substrate onsaid solid phase, or via reactant or substrate present in first flowthat flows into said solid phase where detection occurs.
 83. Method ofclaim 58 wherein said first conjugate binding partner is in the sampledelivery channel.
 84. Method of claim 58 wherein said first conjugatesoluble binding partner is added to said sample before adding to device.85. Method of claim 58 wherein said solid phase is an electrochemicaldetector.
 86. An assay method for the determination of an analyte in aliquid sample comprising the steps of: (a) forming a mixture of a samplecontaining said analyte with: (i) a first immunological binding partnerto said analyte, wherein the first immunological binding partnercontains a detectable label, and (ii) a second immunological bindingpartner of said analyte which is nondiffusively immobilized on a solidphase; (b) allowing the first and second immunological binding partnerto form an immune complex with said analyte, wherein said solid phase incontact with a second fluid path material initially impermeable to saidsample, and which defines a first flow path; (c) flowing a wash reagent,or wash/substrate reagent containing a surface active agent along asecond flow path, within said second fluid path material, such that thesurface active agent allows the wash reagent to flow through said secondflow path, thereby removing unbound reactants from said solid phase; (d)detecting the label attached to the first conjugate binding partner aspart of the immune complex formed in (b).
 87. Method of claim 86 whereinthe detecting step (d) is by a flowing a substrate reagent through saidsecond flow path in said second fluid path material.
 88. Method of claim86 wherein said reagent permeable to said second fluid path material isa wash solution containing a surface active agent.
 89. Method of claim86 wherein said surface active agent is a surfactant, alcohol, or anyother miscible organic solvent.
 90. Method of claim 86 wherein saidsecond fluid path material is a bibulous material.
 91. Method of claim90 wherein said bibulous material is a non-woven polyester fibermaterial.
 92. Method of claim 86 wherein said second fluid path materialis plastic.
 93. Method of claim 86 wherein said second fluid pathmaterial is a structure formed from a hydrophobic polymer.
 94. Method ofclaim 86 wherein said solid phase is a formed (molded or cast) open porestructure.
 95. Method of claim 94 wherein said open pore structure isnylon.
 96. Method of claim 95 wherein said open pore structure isnitrocellulose.
 97. Method of claim 86 wherein said solid phase is aparticulate material.
 98. Method of claim 97 wherein said solid phase isglass particles.
 99. Method of claim 97 wherein said solid phase ispolymer particles.
 100. Method of claim 86 wherein said detectable labelis a light absorbing particle.
 101. Method of claim 100 wherein saidlight absorbing particle is colloidal gold.
 102. Method of claim 100wherein said light absorbing particle is a colored latex particle. 103.Method of claim 86 wherein said detectable label is a phosphorescentmolecule.
 104. Method of claim 86 wherein said detectable label is afluorescent molecule.
 105. Method of claim 86 wherein said detectablelabel is an enzyme.
 106. Method of claim 105 wherein said enzyme ishorseradish peroxidase.
 107. Method of claim 86 wherein said receptormolecule covalently attached to said second conjugate binding partner isbiotin.
 108. Method of claim 107 wherein said receptor-binding partnernon-diffusively bound to said solid phase is avidin.
 109. Method ofclaim 86 wherein detection occurs via immobilized reactant or substrateon said solid phase, or via reactant or substrate present in first flowpath that flows into said solid phase where detection occurs. 110.Method of claim 86 wherein said first immunological binding partner isin the sample delivery channel.
 111. Method of claim 86 wherein saidfirst immunological binding partner is added to the sample before addingsaid sample to the device.
 112. Method of claim 86 wherein said solidphase is an electrochemical detector.
 113. An assay method for thedetermination of an analyte in a liquid sample comprising the steps of:(a) forming a mixture of a sample and a first immunologcal bindingpartner to said analyte, wherein said first immunological bindingpartner contains a detectable label; (b) allowing said first conjugatesoluble binding partner to form a complex with said analyte; (c)contacting said complex with a second immunological binding partner forsaid analyte which is non-diffusively immobilized on a solid phasewherein said solid phase in contact with a second fluid path materialinitially impermeable to said sample, and which defines a first flowpath; (d) flowing a wash solution containing a surface active agentalong a second flow path, within said second fluid path material, suchthat the surface active agent allows the said wash reagent to flowthrough said second flow path, thereby removing unbound reactants fromsaid solid phase; (e) detecting the label attached to the firstconjugate binding partner as part of the immune complex formed in (b).114. Method of claim 113 wherein the detecting step (e) is by flowing asubstrate reagent through said second flow path in said second fluidpath material.
 115. Method of claim 114 wherein said substrate reagentis a substrate for an enzyme.
 116. Method of claim 113 wherein saidreagent permeable to said second fluid path material is a wash solutioncontaining a surface active agent.
 117. Method of claim 116 wherein saidsurface active agent is a surfactant, alcohol, or any other miscibleorganic solvent.
 118. Method of claim 113 wherein said second fluid pathmaterial is a bibulous material.
 119. Method of claim 118 wherein saidbibulous material is a non-woven polyester fiber material.
 120. Methodof claim 113 wherein said second fluid path material is plastic. 121.Method of claim 113 wherein said second fluid path material is astructure formed from a hydrophobic polymer.
 122. Method of claim 113wherein said solid phase is a formed (molded or cast) open porestructure.
 123. Method of claim 122 wherein said open pore structure isnylon.
 124. Method of claim 122 wherein said open pore structure isnitrocellulose.
 125. Method of claim 113 wherein said solid phase is aparticulate material.
 126. Method of claim 125 wherein said solid phaseis glass particles.
 127. Method of claim 125 wherein said solid phase ispolymer particles.
 128. Method of claim 113 wherein said detectablelabel is a light absorbing particle.
 129. Method of claim 128 whereinsaid light absorbing particle is colloidal gold.
 130. Method of claim128 wherein said light absorbing particle is a colored latex particle.131. Method of claim 113 wherein said detectable label is aphosphorescent molecule.
 132. Method of claim 113 wherein saiddetectable label is a fluorescent molecule.
 133. Method of claim 113wherein said detectable label is an enzyme.
 134. Method of claim 113wherein said enzyme is horseradish peroxidase.
 135. Method of claim 113wherein said receptor molecule covalently attached to said secondconjugate binding partner is biotin.
 136. Method of claim 135 whereinsaid receptor-binding partner non-diffusively bound to said solid phaseis avidin.
 137. Method of claim 113 wherein detection occurs viaimmobilized reactant or substrate on said solid phase, or via reactantor substrate present in first flow path that flows into said solid phasewhere detection occurs.
 138. Method of claim 113 wherein firstimmunological binding partner is in the sample delivery channel. 139.Method of claim 113 wherein said first immunological binding partner isadded to sample before adding it to device.
 140. Method of claim 113wherein said solid phase is an electrochemical detector.
 141. An assaymethod for the determination of an analyte in a liquid sample comprisingthe steps of: (a) forming a mixture of a sample containing the substancewith: (i) a first conjugate soluble binding partner to the substance,wherein the first conjugate soluble binding partner contains adetectable label, (ii) a second conjugate soluble binding partner of thesubstance wherein the second soluble conjugate binding partner has beenmodified by the covalent attachment of a receptor molecule, and (iii) asolid phase material wherein a binding partner of the receptor moleculeattached to the second conjugate soluble binding partner has beennon-diffusively bound to the solid phase material; (b) simultaneouslyallowing the first and second conjugate soluble binding partners to forman immune complex with said analyte, and the receptor molecule attachedto the second conjugate soluble binding partner and the receptor-bindingpartner non-diffusively bound to the solid phase to form an immobilizedcomplex, wherein said solid phase is in contact with a second fluid pathmaterial initially impermeable to said mixture, and which defines afirst flow path; (c) flowing a wash solution containing a surface activeagent along a second flow path, within said second fluid path material,such that the surface active agent allows the wash reagent to flowthrough said second flow path, thereby removing unbound reactants fromsaid solid phase; (d) detecting the label attached to the firstconjugate binding partner as part of the immune and immobilizedcomplexes formed in (b).
 142. Method of claim 141 wherein the detectingstep (d) is by a flowing a substrate reagent through said second flowpath in said second fluid path material.
 143. Method of claim 142wherein said substrate reagent is a substrate for an enzyme.
 144. Methodof claim 141 wherein said reagent permeable to said second fluid pathmaterial is a wash solution containing a surface active agent. 145.Method of claim 141 wherein said surface active agent is a surfactant,alcohol, or any other miscible organic solvent.
 146. Method of claim 141wherein said second fluid path material is a bibulous material. 147.Method of claim 146 wherein said bibulous material is a non-wovenpolyester fiber material.
 148. Method of claim 141 wherein said secondfluid path material is plastic.
 149. Method of claim 141 wherein saidsecond fluid path material is a structure formed from a hydrophobicpolymer.
 150. Method of claim 141 wherein said solid phase is a formed(molded or cast) open pore structure.
 151. Method of claim 150 whereinsaid open pore structure is nylon.
 152. Method of claim 150 wherein saidopen pore structure is nitrocellulose.
 153. Method of claim 141 whereinsaid solid phase is a particulate material.
 154. Method of claim 153wherein said solid phase is glass particles.
 155. Method of claim 153wherein said solid phase is polymer particles.
 156. Method of claim 141wherein said detectable label is a light absorbing particle.
 157. Methodof claim 156 wherein said light absorbing particle is colloidal gold.158. Method of claim 156 wherein said light absorbing particle is acolored latex particle.
 159. Method of claim 141 wherein said detectablelabel is a phosphorescent molecule.
 160. Method of claim 141 whereinsaid detectable label is a fluorescent molecule.
 161. Method of claim141 wherein said detectable label is an enzyme.
 162. Method of claim 161wherein said enzyme is horseradish peroxidase.
 163. Method of claim 141wherein said receptor molecule covalently attached to said secondconjugate binding partner is biotin.
 164. Method of claim 163 whereinsaid receptor-binding partner non-diffusively bound to said solid phaseis avidin.
 165. Method of claim 141 wherein detection occurs immobilizedreactant or substrate on said solid phase, or via reactant or substratepresent in first flow path that flows into said solid phase wheredetection occurs.
 166. Method of claim 141 wherein said first conjugatesoluble binding reagent is added to sample delivery channel.
 167. Methodof claim 141 wherein said first conjugate soluble binding reagent isadded to said sample before adding it to the device.
 168. Method ofclaim 141 wherein said solid phase is an electrochemical detector. 169.An assay method for the determination of a analyte in a liquid samplecomprising the steps of: (a) forming a mixture of a sample and a firstbinding partner to the analyte, wherein said first binding partnercontains a detectable label; (b) allowing said first conjugate solublebinding partner to form a complex with said analyte; (c) contacting saidcomplex with the analyte or an analog of the analyte which isnondiffusively immobilized on a solid phase wherein said solid phase incontact with a second fluid path material initially impermeable to saidsample, and which defines a first flow path; (d) flowing a wash solutioncontaining a surface active agent along a second flow path, within saidsecond fluid path material, such that the surface active agent allowsthe said wash reagent to flow through said second flow path, therebyremoving unbound reactants from said solid phase; (e) detecting thelabel attached to the solid phase, and relating inhibition of the amountof label to the concentration of the analyte.
 170. An assay method forthe determination of a analyte in a liquid sample comprising the stepsof: (a) forming a mixture of a sample and a first binding partner to theanalyte, wherein said first binding partner contains a detectable label,and a second reagent comprising the analyte or an analyte analogcovalently coupled to a receptor molecule; (b) allowing said firstconjugate soluble binding partner to form complexes with said analyteand said analyte/analyte analog/receptor conjugate; (c) contacting saidmixture with a binding partner of the receptor molecule which isnondiffusively immobilized on a solid phase wherein said solid phase incontact with a second fluid path material initially impermeable to saidsample, and which defines a first flow path; (d) flowing a wash solutioncontaining a surface active agent along a second flow path, within saidsecond fluid path material, such that the surface active agent allowsthe said wash reagent to flow through said second flow path, therebyremoving unbound reactants from said solid phase; (e) detecting thelabel attached to the solid phase, and relating inhibition of the amountof label to the concentration of the analyte.
 171. An assay method forthe determination of a analyte in a liquid sample comprising the stepsof: (a) forming a mixture of a sample and a conjugate of the analyte oran analog of the analyte bound to a detectable label; (b) allowing saidfirst conjugate soluble binding partner to form a complex with saidanalyte; (c) contacting said mixture with a binding partner of theanalyte which is nondiffusively immobilized on a solid phase whereinsaid solid phase in contact with a second fluid path material initiallyimpermeable to said sample, and which defines a first flow path; (d)flowing a wash solution containing a surface active agent along a secondflow path, within said second fluid path material, such that the surfaceactive agent allows the said wash reagent to flow through said secondflow path, thereby removing unbound reactants from said solid phase; (e)detecting the label attached to the solid phase, and relating inhibitionof the amount of label to the concentration of the analyte.
 172. Anassay method for the determination of a analyte in a liquid samplecomprising the steps of: (a) forming a mixture of a sample and aconjugate of the analyte or an analog of the analyte bound to adetectable label, and a second reagent comprising a soluble bindingpartner to the analyte covalently coupled to a receptor molecule; (b)allowing said soluble binding partner of the analyte to form complexeswith said analyte and with said analyte/analyte analog/receptorconjugate; (c) contacting said mixture with a binding partner of thereceptor molecule which is nondiffusively immobilized on a solid phasewherein said solid phase in contact with a second fluid path materialinitially impermeable to said sample, and which defines a first flowpath; (d) flowing a wash solution containing a surface active agentalong a second flow path, within said second fluid path material, suchthat the surface active agent allows the said wash reagent to flowthrough said second flow path, thereby removing unbound reactants fromsaid solid phase; (e) detecting the label attached to the solid phase,and relating inhibition of the amount of label to the concentration ofthe analyte.